The next generation of molecular cancer therapeutics will target pivotal protein-protein interaction interfaces participating in immune cell receptor signaling, oncogenes, and suppressor genes. We will implement a truly transformative, wholly novel, technology ?protein painting? for the rapid direct sequencing of hidden native protein-protein interaction hot spots, that was originated under NCI R21 IMAT CA177535. Our technology employs previously unexplored small molecule (12 ) aryl hydrocarbon dyes or ?paints? to cut out, and MS sequence, only the hidden unmodified contact interfaces between two or more interacting native proteins. Protein painting employs a completely new principle that yields a much higher specificity and three times higher number of positive hits compared to chemical footprinting methods. Paint chemistries have extremely high affinities (rapid on-rates, and very slow off-rates that are ten to 100 times higher than most protein-protein interactions). When mixed with a native pre-formed protein complex for only 5 minutes, the paints non- covalently coat all external sites on the protein without altering the 3D conformation of the complex, but cannot gain access to the solvent inaccessible hidden protein-protein interaction domains. Each paint molecule spans 3 amino acids or less, and has high affinity for protease cleavage consensus sites. Following painting, the unbound paints are washed away and the proteins are dissociated. This leaves the paint molecules coating surfaces not participating in the interface. Following dissociation, the proteins are linearized, digested with proteolytic enzymes, and sequenced by MS. The paint molecules remain non-covalently bound after the proteins are denatured. Proteolytic enzymes such as trypsin will not cleave the regions of the protein that are ?painted?. Following proteolysis, therefore, peptides emerging from MS will exclusively be generated from the unmodified opposing points where the proteins were in intimate contact. All of the original R21 Aims and Milestones were fully met. A very high correlation (p<0.0003) was found between protein painting and the contact points predicted by crystal structure, with a 97% specificity for true positive hot spots. We discovered evolutionarily conserved 3-way hotspots and verified their functional importance by creating peptides and mAbs that block the interaction and extinguish signaling. Under Aim1, we will discover novel hotspot sequences of broad therapeutic relevance for interacting protein complexes of three types: 1) cell surface receptors (PD1/PDL-1,PDL-2) involved in tumor immune cell suppression , 2) Hippo cancer suppressor pathway proteins, and 3) the recently elucidated oncogene Folliculin.Under Aim 2 we will validate the functional (drug candidate) importance of the interaction hotspots we discover, by creating peptides and monoclonal antibodies that bind to the opposing hotspot faces of the interacting proteins. We will verify that these ligands will suppress protein complex formation. For the PD1/PDL-1,PDL-2 cell surface complexes, we will extend protein painting, for the first time, to MS sequence cell-cell interaction contact points in cultured live cells.